Small Schiff Base Molecules—A Possible Strategy to Combat Biofilm-Related Infections

Microorganisms participating in the development of biofilms exhibit heightened resistance to antibiotic treatment, therefore infections involving biofilms have become a problem in recent years as they are more difficult to treat. Consequently, research efforts are directed towards identifying novel molecules that not only possess antimicrobial properties but also demonstrate efficacy against biofilms. While numerous investigations have focused on antimicrobial capabilities of Schiff bases, their potential as antibiofilm agents remains largely unexplored. Thus, the objective of this article is to present a comprehensive overview of the existing scientific literature pertaining to small molecules categorized as Schiff bases with antibiofilm properties. The survey involved querying four databases (Web of Science, ScienceDirect, Scopus and Reaxys). Relevant articles published in the last 10 years were selected and categorized based on the molecular structure into two groups: classical Schiff bases and oximes and hydrazones. Despite the majority of studies indicating a moderate antibiofilm potential of Schiff bases, certain compounds exhibited a noteworthy effect, underscoring the significance of considering this type of molecular modeling when seeking to develop new molecules with antibiofilm effects.


Introduction
Clinically relevant microbial biofilms are defined as "aggregated microbial cells surrounded by a polymeric self-produced matrix, which may contain host components", suspended or attached to a surface [1].Biofilm-related infections attracted the attention of scientists 50 years ago in the context of cystic fibrosis, and their impact on the medical field has grown ever since [2].These infections may be tissue-related (chronic otitis media, chronic sinusitis, chronic laryngitis, dental plaque, endocarditis, cystic fibrosis, kidney stones, biliary tract infections, urinary tract infections, osteomyelitis, wound infections, etc.) or associated with medical devices (contact lenses, endotracheal tubes, cardiac devices or catheters) [1,3].Some examples of biofilm forming pathogens are: Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, Staphylococcus epidermidis, Streptococci, Enterococci and Candida spp.[4].
Biofilm formation requires four stages: (i) attachment of the mobile microorganism to a surface, (ii) colonization, (iii) development and maturation of biofilm and (iv) dispersion and propagation [5,6].Attachment is mediated by cilli, flagella, surface proteins of microorganisms and rugosity of the surface [7].It is reversible at first and then becomes irreversible, triggering transcription of specific genes for signalling molecules and extracellular polymeric substances (EPS).Colonization involves growth and division processes and EPS synthesis [5].A mature biofilm consists of three layers: the biofilm nucleus, membranes of basal microorganisms and external mobile planktonic cells.It is a complex mixture of water, microbial cells, proteins, aminoacids and polysaccharides [6].Dispersion is mediated by external factors or by self-digestion and contributes to dissemination of infection [7].There are two important features of biofilm (sessile) growth compared with the free-floating (planktonic) state that contributes to pathogenicity: increased tolerance to antibiotic treatment and persistence in the host, despite inflammation and immune response [1].The major consequence is that biofilm infections are hard to treat and usually become chronic [8].
Antibiotherapy is active on planktonic microbial cells, but its effectiveness against sessile states is variable, as established biofilms are usually recalcitrant to conventional antibiotics [9].Treatment may require higher doses of antimicrobials, prolonged duration [8,10], combination therapy [11,12] or special modes of administration (nebulized antibiotics) [13].
There is a constant need to develop alternative antibiofilm strategies, and extensive research has been conducted in this direction [10].Antibiofilm small molecules are relevant because they target stages in biofilm development which are different to those of normal planktonic state [9].The mechanisms (Figure 1) may involve: blocking microbial adhesion (biocides [14], antibiotics [15] and impregnated coatings [16]), inhibition of microbial communication (quorum sensing inhibitors and quorum quenching) [17] and killing cells inside the biofilm (persisters or non-growing cells) (cisplatin, cis-2-decenoic acid, colistin, mytomicin C) [18][19][20][21].In particular, strategies like quorum sensing inhibition may prove useful because they do not necessarily affect bacterial growth but they reduce virulence, thus increasing susceptibility of microorganisms to antibiotics and to host immune cells without the risk of antibiotic resistant [17].
Antibiotics 2024, 13, x FOR PEER REVIEW 2 of 33 mediated by external factors or by self-digestion and contributes to dissemination of infection [7].There are two important features of biofilm (sessile) growth compared with the free-floating (planktonic) state that contributes to pathogenicity: increased tolerance to antibiotic treatment and persistence in the host, despite inflammation and immune response [1].The major consequence is that biofilm infections are hard to treat and usually become chronic [8].
Antibiotherapy is active on planktonic microbial cells, but its effectiveness against sessile states is variable, as established biofilms are usually recalcitrant to conventional antibiotics [9].Treatment may require higher doses of antimicrobials, prolonged duration [8,10], combination therapy [11,12] or special modes of administration (nebulized antibiotics) [13].
Human and veterinary therapy benefits from several antibacterial drugs recognized as Schiff bases.In multidrug-resistant tuberculosis (MDR-TB), a longer treatment regimen includes two Schiff bases which act on Mycobacterium tuberculosis cell wall: bacteriostatic terizidone (Figure 2), a cycloserine derivative, analogue of D-alanine and anti-leprosy clofazimine, which is an iminophenazine [63,64].
Therefore, this review aims to provide a comprehensive overview of the existing scientific literature on small molecules classified as Schiff bases with antibiofilm properties.Antibiotics 2024, 13 Numerous reviews have explored the antimicrobial potential of Schiff bases [88][89][90], as well as Schiff base-derived metal complexes [89,[91][92][93], nanoparticles and modified chitosan [90,94].While there are reports on the antibiofilm potential of Schiff base metal complexes [95,96], there is limited information on small molecules.
Therefore, this review aims to provide a comprehensive overview of the existing scientific literature on small molecules classified as Schiff bases with antibiofilm properties.

Results
The literature survey is summarized in Table 1.The relevant articles were selected and divided into two categories based on structure: classical Schiff bases and oximes and hydrazones.

Compounds
Biological Assay/Microorganism Observations Ref.

Classical Schiff Bases
Antibiotics 2024, 13, x FOR PEER REVIEW 5 of 33

Results
The literature survey is summarized in Table 1.The relevant articles were selected and divided into two categories based on structure: classical Schiff bases and oximes and hydrazones.

Results
The literature survey is summarized in Table 1.The relevant articles were selected and divided into two categories based on structure: classical Schiff bases and oximes and hydrazones.

Discussion
Due to their ease of synthesis and their wide range of applications, salicylaldehyde Schiff bases are frequently cited in the relevant literature.These compounds demonstrate antimicrobial potential, both as simple ligands and as metal complexes [56][57][58].The antimicrobial activity is directly influenced by substitutions on the salicyl moiety, with halogenation exerting a noticeable impact in particular [58].
p-Aminobenzoic acid (PABA) is an amino acid derivative, implicated in folate biosynthesis in microbial cells [133].Due to its importance for bacterial viability, it serves as a target for antimicrobial therapy [134].Therefore, obtaining hybrid molecules is a direction of molecular development [135] in the search for new anti-infective agents.
Starting from a series of Schiff base derivatives of p-aminobenzoic acid and halogenated salicylaldehydes (compound 1), me-too analogues were synthesized and tested for antimicrobial, antibiofilm and cytotoxicity activities [99].The design approaches were as

Discussion
Due to their ease of synthesis and their wide range of applications, salicylaldehyde Schiff bases are frequently cited in the relevant literature.These compounds demonstrate antimicrobial potential, both as simple ligands and as metal complexes [56][57][58].The antimicrobial activity is directly influenced by substitutions on the salicyl moiety, with halogenation exerting a noticeable impact in particular [58].
p-Aminobenzoic acid (PABA) is an amino acid derivative, implicated in folate biosynthesis in microbial cells [133].Due to its importance for bacterial viability, it serves as a target for antimicrobial therapy [134].Therefore, obtaining hybrid molecules is a direction of molecular development [135] in the search for new anti-infective agents.

Discussion
Due to their ease of synthesis and their wide range of applications, salicylaldehyde Schiff bases are frequently cited in the relevant literature.These compounds demonstrate antimicrobial potential, both as simple ligands and as metal complexes [56][57][58].The antimicrobial activity is directly influenced by substitutions on the salicyl moiety, with halogenation exerting a noticeable impact in particular [58].
p-Aminobenzoic acid (PABA) is an amino acid derivative, implicated in folate biosynthesis in microbial cells [133].Due to its importance for bacterial viability, it serves as a target for antimicrobial therapy [134].Therefore, obtaining hybrid molecules is a direction of molecular development [135] in the search for new anti-infective agents.
Bacterial fatty acid synthetase may serve as the target for the development of new antibacterial agents.Triclosan and other 2-hydroxydiphenyl ethers demonstrated inhibition against enoyl-acyl carrier protein reductase (FabI), a key enzyme in fatty acid production [138,139].Schiff bases and hydrazones have also been reported as inhibitors of staphylococcal β-ketoacyl carrier proteinsynthase III (encoded by FabH gene) [140,141].In Gram-negative bacteria, PqsD-an enzyme implicated in Pseudomonas autoinductors synthesis-is structurally related to FabH.Thus, inhibitors of fatty acid synthetases may also act against PqsD [142].
A series of hybrid Schiff bases (21a-f) were synthesized by incorporating isatin, pyrazole and either piperidin-1ylsulfonyl or N-methylpiperazin-1ylsulfonyl into a single molecule [111].These resultant molecules are amphiphilic in nature, stemming from exhibited quorum sensing inhibition against C. violaceum and was able to reduce swarming motility of P. aeruginosa by 14.4-45.7%at MIC/4-MIC.Thus, it targets two steps in biofilm formation: communication and dispersion [115].
5-Nitro-2-thiophenecarbaldehyde N-[(E)-(5-nitrothienyl)methylidene)hydrazone (40) was evaluated for antistaphylococcal activity [128].It inhibited Pan-S S. aureus at 0.5-2.0µg/mL, VRSA and MRSA.Exposing the biofilm to this compound for 24 h led to a noteworthy (p < 0.05) decrease in the integrity of S. aureus biofilm at a concentration 4× MIC.The findings indicate that this hydrazone can impact S. aureus biofilm integrity even at concentrations 10-40× MIC.Additional research is necessary to gain a deeper understanding of the mechanism behind the disruption of S. aureus biofilm and potential interactions with biofilm-targeting properties of 40 and other antimicrobials available in clinical settings.

Materials and Methods
The literature survey was conducted across four databases (Web of Science, Sci-enceDirect, Scopus and Reaxys).The primary keywords employed were ("imine" OR "azomethine" OR "Schiff base" OR "hydrazone" OR "oxime") AND ("biofilm" OR "biofilm inhibitor" OR "antibiofilm" OR "anti-biofilm"), covering a ten-year span (2013-2023) with search parameters adjusted for each database.Inclusion criteria comprised of the English language, original research articles, antibiofilm evaluation and a focus on small molecules.The emphasis was on Schiff bases acting as antimicrobials and antibiofilm agents with medical applications, leading to the exclusion of metal complexes, Schiff base polymers, antibiofouling agents and other categories.It is noteworthy that while the scientific literature extensively covers antibacterial evaluations of Schiff bases, only a limited number of studies tested their antibiofilm potential.Consequently, numerous articles had to be excluded.Following duplication removal, title and abstract screening, full-text screening, 2-Methyl-l1-hydroxyimino-6,11-dihydrodibenzo[b,e]thiepin-5,5-dioxide (44a) and 2methyl-l1-hydroxyimino-6,11-dihydrodibenzo[b,e]thiepin-5,5-dioxide (44b) [131] demonstrated microbicidal activity against the Gram-negative, non-fermentative A. baumanii.These oximes effectively hindered the adherence ability of C. albicans strains to inert substrata at a concentration of 250 µg/mL.Additionally, they displayed notable antibiofilm activity against the Gram-negative, non-fermentative bacilli P. aeruginosa and A. baumanii.Molecular modelling suggests that these compounds may interfere with the synthesis of quorum sensing molecules, specifically N-acyl-l-homoserine lactones, utilized by Gramnegative strains as their potential targets.It's worth noting that despite the absence of fungicidal activity, compounds 44a and 44b exhibited inhibitory effects on the development of fungal biofilms.

Materials and Methods
The literature survey was conducted across four databases (Web of Science, ScienceDirect, Scopus and Reaxys).The primary keywords employed were ("imine" OR "azomethine" OR "Schiff base" OR "hydrazone" OR "oxime") AND ("biofilm" OR "biofilm inhibitor" OR "antibiofilm" OR "anti-biofilm"), covering a ten-year span (2013-2023) with search parameters adjusted for each database.Inclusion criteria comprised of the English language, original research articles, antibiofilm evaluation and a focus on small molecules.The emphasis was on Schiff bases acting as antimicrobials and antibiofilm agents with medical applications, leading to the exclusion of metal complexes, Schiff base polymers, antibiofouling agents and other categories.It is noteworthy that while the scientific literature extensively covers antibacterial evaluations of Schiff bases, only a limited number of studies tested their antibiofilm potential.Consequently, numerous articles had to be excluded.Following duplication removal, title and abstract screening, full-text screening, eligibility analysis and cross-checking, the most relevant articles were selected and reviewed.

Conclusions
Although Schiff bases have demonstrated their antimicrobial efficacy, their potential as small molecules to inhibit biofilm formation remains an area that requires further exploration.A significant challenge is that while the antimicrobial screening is a routine practice for assessing the biological potential of Schiff bases, antibiofilm assays are not consistently taken into consideration.
Imine moiety may be included in a molecule for several reasons.For example, it may serve as a link between two structures resulting in hybrid compounds.It may also be used in isostere substitution of carbonyl group-amide or ether.Imine groups are polar and may form hydrogen bonds with aminoacids from the active sites as they are important for biological activity.
There are several cases where the antimicrobial activity of Schiff bases and their derivatives were superior compared to the parent compound, even turning bacteriostatic action into bactericidal.The spectrum may be reduced to Gram-positive like Staphylococcus aureus for halogenated salicylaldehyde Schiff bases or Enterococcus faecalis for benzensulfonyl thiazoloimines.Some Schiff bases have a broad spectrum, including Gram-negatives such as P. aeruginosa, K. pneumoniae, A. baumannii and fungi.
The antibiofilm potential is variable and usually moderate compared to antimicrobial activity of the same compounds or references.In general, the most active antimicrobials were evaluated for biofilm inhibition.There are also Schiff bases, as we have shown in this article, which presented a remarkably biofilm inhibition.
According to the findings outlined in this article, it can be confirmed that Schiff bases serve as a molecular framework worthy of investigation for their potential antibiofilm properties.The prospective areas for future research include synthesizing hybrid compounds within this class, utilizing known antimicrobial agents as starting materials and testing the antibiofilm efficacy of certain Schiff bases recognized for different therapeutic applications, which has aims to reposition these substances for antibiofilm purposes.Furthermore, Schiff bases with antimicrobial and antibiofilm properties, originating from natural products, present a promising avenue for identifying potential lead molecules.Finally, it is crucial to conduct further studies on the Schiff bases that have demonstrated antibiofilm efficacy.These investigations should focus on unraveling their mechanisms of action, exploring potential synergistic relationships, assessing therapeutic potency and ensuring safety.
Thus, Schiff bases still remain an open door for the antibiofilm research.

Figure 1 .
Figure 1.Antibiofilm mechanisms of action for small molecules.

Figure 1 .
Figure 1.Antibiofilm mechanisms of action for small molecules.